Method of Diagnosing Sjogren&#39;s Disease

ABSTRACT

Provided are methods and compositions for determining whether an individual has Sjögren&#39;s disease (SD). The method entails determining in a biological sample from the individual the presence of antibodies directed to salivary gland protein 1 (SP-I), parotid secretory protein (PSP), carbonic anhydrase 6 (C A6), or determining a combination of the antibodies. Determining that the individual has SD is based on the presence of the antibodies. The method provides for detection of early SD. Kits for antibody detection containing the antigens to which the antibodies of SD patients are directed are also provided.

This application claims priority to U.S. provisional application no.61/297,167, filed Jan. 21, 2010, the disclosure of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Sjogren's disease is a common autoimmune disorder with significantmorbidity and mortality secondary to destruction of the salivary andlachrymal glands. It involves both local and systemic autoimmunity andis generally recognized only after salivary and lachrymal glands aredestroyed resulting in dry mouth and dry eyes (Borchers, A. T., S. M.Naguwa, C. L. Keen, and M. E. Gershwin. 2003. Immunopathogenesis ofSjogren's syndrome. Clin Rev Allergy Immunol 25:89-104; Delaleu, N., R.Jonsson, and M. M. Koller. 2005. Sjogren's syndrome. Eur J Oral Sci113:101-113). Sjögren's syndrome is a common syndrome affecting 0.5% ofthe population with a strong female predominance (Delaleu, N., R.Jonsson, and M. M. Koller. 2005. Sjogren's syndrome. Eur J Oral Sci113:101-113, Fox, R. I. 2005. Sjogren's syndrome. Lancet 366:321-331.).It consists of xerostomia and xerophthalmia and may be due to severalcauses including aging, infections, medications, environmental toxinsand autoimmune responses (Daniels, T. E. 2000. Evaluation, differentialdiagnosis, and treatment of xerostomia. J Rheumatol Suppl 61:6-10).Sjogren's disease is a primary disorder consisting of Sjogren's syndromewith systemic manifestations including lymphadenopathy, interstitialpneumonitis and mild renal disease (Borchers, A. T., S. M. Naguwa, C. L.Keen, and M. E. Gershwin. 2003. Immunopathogenesis of Sjogren'ssyndrome. Clin Rev Allergy Immunol 25:89-104; Delaleu, N., R. Jonsson,and M. M. Koller. 2005. Sjogren's syndrome. Eur J Oral Sci 113:101-113).Sjogren's syndrome is often seen in association with other autoimmunediseases, especially systemic lupus erythematosus (SLE) (Manoussakis, M.N., et al. Moutsopoulos. 2004. Sjogren's syndrome associated withsystemic lupus erythematosus: clinical and laboratory profiles andcomparison with primary Sjogren's syndrome. Arthritis Rheum 50:882-891).Patients with Sjogren's disease often have hypergammaglobulinemia, andvarious autoantibodies, especially to Ro and La (Fox, R. I. 2005.Sjogren's syndrome. Lancet 366:321-331, Lazarus, M. N., and D. A.Isenberg. 2005. Development of additional autoimmune diseases in apopulation of patients with primary Sjogren's syndrome. Ann Rheum Dis64:1062-1064, ansen, A., P. E. Lipsky, and T. Dorner. 2005.Immunopathogenesis of primary Sjogren's syndrome: implications fordisease management and therapy. Curr Opin Rheumatol 17:558-565). Almost4% of patients with Sjögren's disease will develop lymphoma,predominantly B cell non-Hodgkin lymphomas.

The diagnosis of Sjogren's disease is generally made when dry eyescauses irritation and corneal abrasions, and dry mouth causes difficultyswallowing and dental caries. Biochemical diagnosis is based ondetection of lymphocytes infiltrating the salivary glands and serum autoantibodies directed towards Ro and La. Current therapies involve the useof artificial tears and saliva as well as cholinergic drugs to enhancesecretions from the few remaining glandular cells (the disclosure of thefollowing three citations are incorporated herein by reference: Kassan,S. S., and H. M. Moutsopoulos. 2004. Clinical manifestations and earlydiagnosis of Sjogren syndrome. Arch Intern Med 164:1275-1284, Latkany,R. 2008. Dry eyes: etiology and management. Current Opinion inOphthalmology 19:287-291, Thanou-Stavraki, A., and J. A. James. 2008.Primary Sjogren's syndrome: Current and prospective therapies. Seminarsin Arthritis and Rheumatism 37:273-292). However, no current therapiesrestore salivary and lachrymal gland function because the glands arelargely destroyed by the time the disease is identified. It wouldtherefore be of great benefit to be able to diagnose Sjogren's diseaseearly since that is when it is amenable for treatment, but no suchdiagnostic methods exist. Thus, there is an ongoing and unmet need forimproved methods for diagnosing Sjogren's disease, and in particular foruse in diagnosis before the diseases progresses to a point where currenttherapeutic approaches are inadequate.

SUMMARY OF THE INVENTION

The present invention provides a method for determining whether anindividual has Sjogren's disease (SD). The method comprises determiningin a biological sample from the individual the presence of antibodiesdirected to salivary gland protein 1 (SP-1), parotid secretory protein(PSP), carbonic anhydrase 6 (CA6), or determining a combination of theantibodies. Determining that the individual has SD is based on thepresence of the antibodies. Also provided is a method for determiningthat an individual does not have SD which comprises determining in abiological sample obtained from the individual the absence of detectionof antibodies to PSP, and SP-1 and determining based on the absence ofdetection of the antibodies that the individual does not have SD. Theindividual may also have less antibodies to CA6 relative to an SDpatient. Any PSP or CA6 protein may be used. However, there is no knownhuman homologue to SP-1, and the invention accordingly provides a noveland unexpected discovery that humans with SD produce autoantibodies thatrecognize non-human SP-1, and in particular murine SP-1. It is expectedthat SP-1 produced in other non-human mammals can also be used fordetecting anti-SP-1 antibodies.

The method of the invention can be used to diagnose SD in any individualof any age or gender, and at any stage of the disease. In oneembodiment, the invention is used to detect early SD.

The antibodies that are positively associated with SD and describedfurther herein can be detected using any suitable method and/or reagentsfor detecting antibodies. In one embodiment, the antibodies are detectedusing an ELISA assay.

The invention also provides kits comprising the antigens to which theantibodies of SD patients are directed and may further comprisecomponents for biological sample collection, reagents for antibodydetection, control reaction, and other materials useful for detectingantibodies. In one embodiment, the invention provides a kit comprisingpurified SP-1, PSP and CA6 proteins or fragments thereof that arerecognized by antibodies produced by SD patients. The proteins may beprovided in isolated and purified form, and they may be covalently ornon-covalently associated with a solid matrix.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a photographic representation of Western blotting forautoantibodies in the sera of IL-14aTG mice during early stages ofSjogren's Disease. To obtain the data summarized in FIG. 1, sera werecollected from IL-14aTG mice, NOD and C57BL/6 mice at 7 months of age.Custom expressed and purified salivary gland protein 1 (SP-1), parotidsecretory protein (PSP) and carbonic anhydrase 6 (CA6) were used to runWestern blots with these sera. Data shown are representative of 6 micestudied in each group. The left panel shows the serum of an IL-14aTGmouse that recognizes CA6 and PSP strongly and SP-1 weakly. The middlepanel shows the same study with the serum of a C57BL/6 mouse. C57BL/6sera bound none of these auto-antigens. The right panel shows the samestudy with the serum of an NOD mouse. Both CA6 and PSP are stronglyrecognized by this serum.

FIG. 2 provides a photographic representation of Western blottingresults demonstrating that lymphotoxin is found in the salivary glandsecretions of IL-14a TG mice but not littermate controls.

FIG. 3 provides a graphical representation of data showing thatlymphotoxin is found in the sera of selected patients with Sjogren'sdisease.

FIG. 4 provides a photographic representation of Western blottingshowing autoantibodies in the sera and salivary glands ofIL-14aTG.LTA−/− mice. The data show that the serum of an IL-14aTG.LTA−/−mouse at 11 months of age reacts with CA6 and PSP.

FIG. 5 provides a photographic representation of Western blottingresults showing that sera from Patients with Sjogren's Disease ContainAutoantibodies to CA6, PSP and SP-1. The Western blots were performed asin FIG. 1 except sera were used from patients with Sjogren's disease oraged matched normal controls. Five patients and two normal controls wereevaluated. Data shown are from one representative patient (all fivepatients showed similar results) and one normal control (both normalcontrols showed similar results). Patients with SD but not normalcontrols have antibodies to PSP and SP-1.

DESCRIPTION OF THE INVENTION

The present invention provides a method for determining whether anindividual has Sjogren's disease (SD). The method comprises determiningin a biological sample obtained from the individual the presence ofantibodies to salivary gland protein 1 (SP-1), parotid secretory protein(PSP), carbonic anhydrase 6 (CA6), or a combination of the SP-1, PSP andCA6 antibodies. The individual is identified as having SD if suchantibodies or combinations thereof are present.

The invention provides the first identification of a positivecorrelation between SD and antibodies to any of SP-1, PSP and CA6. Ourdiscovery that human SD patients produce antibodies that recognizemurine SP-1 was particularly unexpected because there is no known humanhomologue to this protein. Thus, the invention provides a surprising andnovel method for identifying individuals who have SD. The inventionfurther provides for identifying an individual as not having SD ifantibodies to SP-1 and PSP are not detected in a biological sampleobtained from the individual.

In addition to antibodies to SP-1, PSP and CA6, antibodies to othermarkers can be determined in the method of the invention for evaluatingwhether or not an individual has SD. Non-limiting examples of suchantibodies include those directed to lymphotoxin (LTA), mucin 10(submandibular gland salivary mucin), salivary amylase 1, Ro, La,muscarinic receptor 3, fodrin, and the cytokines IL-14 and interferon-α.

Our demonstration that production of antibodies to SP-1, PSP and CA6 areindicative of SD is supplemented by research we performed on clinicallyrelevant animal models of SD. In this regard, we have developed ananimal model that reproduces all the immunological and clinical featuresseen in patients with SD in the same relative time frame. The animalmodel we developed is referred to as the IL-14alpha transgenic mouse(IL14aTG). Using this model we have demonstrated that lymphocyticinfiltration of the salivary glands occurs after the glands have alreadybeen destroyed and that only a small percentage of the mice developantibodies to Ro and La. Our data also demonstrate that LTA is importantto early salivary gland injury in IL14aTG and the NOD mouse models ofSD, and that IL-14aTG mice lacking LTA (IL-14aTG.LTA−/−) retain normalsalivary gland function and suffer no lymphocytic infiltration of theirsalivary glands. We also demonstrate that LTA is found in the serum ofhuman SD patients.

IL-14aTG mice, like patients with SD, produce interferon-α (IFN-a)systemically and lymphotoxin (LTA) locally in the salivary glands.Autoantibodies are deposited in the salivary gland at the time thatsalivary gland function is lost. The auto-antigens recognized at thisstage are different than the auto-antigens seen later in the disease, Roand La, which traditionally have been felt to be characteristic of SD.We have also demonstrated in this model that salivary gland function islost before infiltration of the salivary glands with lymphocytes. Insummary, and without intending to be bound by any particular theory, itis considered that IL-14aTG mice reproduce all the features of SD seenin patients in the same relative time frame. Further, SD occurs in allIL-14aTG mice tested. The time course of SD in IL-14aTG mice is 1)hypergammaglobulinemia and early antibody production at 6 months of age,2) decreased salivary gland function with lymphocytic infiltration ofonly the submandibular glands, but antibody deposition in thesubmandibular and parotid glands at 10 months, 3) lymphocyticinfiltration of the submandibular, parotid and lachrymal glands with Band T cells and plasma cells along with mild renal and lung disease at14 months, and 4) large B cell lymphoma at 18 months. Note that loss ofsalivary gland function occurs several months before lymphocyticinfiltration of the salivary glands, which indicates there is antibodyand/or cytokine mediated injury that occurs before lymphocyticinfiltration of the glands. Furthermore, as noted above, the IL-14aTGmice generally do not produce anti-Ro and anti-La antibodies during theearly stages of the disease. The pattern of immunofluorescence forimmunoglobulin deposition in the salivary glands varies over time,suggesting that different auto antigens are likely to be important atvarious stages of the disease. Additionally, IL-14aTG mice do notdevelop diabetes, like NOD mice, or proliferative glomerulonephritis,like (NZB×NZW) F1 and MRL/1pr mice. The IL-14aTG mouse is thus the onlyanimal model of SD that develops all the features of Sjogren's diseasein the absence of other autoimmune diseases. Accordingly, the IL-14aTGand IL-14aTG.LTA mice are valuable for identifying early events in thedevelopment of SD.

We identified the SD antigens disclosed herein in part by examining theexpression of mRNA in the spleens of IL14aTG mice. We have producedpurified antigens encoded by these mRNAs and have shown that IL14aTG andNOD mouse models of SD develop antibodies to these antigens during theearly course of their disease (FIG. 1). We have also demonstrated thatlymphotoxin is present in the salivary gland secretions of IL14aTG miceduring the early course of their disease, and that elimination oflymphotoxin prevents the development of salivary gland injury (FIG. 2).We have shown that lymphotoxin is present in the sera of human patientswith SD (FIG. 3). Further, we have demonstrated the presence ofantibodies to SD antigens in IL-14aTG.LTA−/− mice (FIG. 4.) Furtherstill, we also demonstrate that sera from human patients with SD containautoantibodies to CA6, PSP and SP-1 (the latter of which as describedabove has no known human homologue) while normal controls do not haveantibodies to PSP-1 or SP-1, and have only weak antibody response to CA6(FIG. 5). The antibody response to CA6 was stronger in SD patients thanin normal controls.

Each of the SD markers to which antibodies are determined according tothe method of the invention are well characterized and are known in theart and their coding and amino acid sequences are available in GenBank.Each GenBank reference presented in this disclosure is incorporatedherein by reference as of the date of this invention. It is expectedthat any mammalian CA6, PSP and SP-1 antigens (with the caveat thatthere is no know human homologue to SP-1) can be used with any of a widevariety of established immunoassays in the method of the invention todetermine whether or not an individual produces antibodies directed tothe antigens. In one embodiment, murine PSP is described by thesequences presented in GenBank entry NM_(—)008953.2. In one embodiment,murine SP-1 described by the sequences presented in GenBank entryNM_(—)009267.2. In one embodiment, CA6 is described by the sequencespresented in GenBank entry NM_(—)009802.2.

The protein antigens that are used for detecting autoantibodiesaccording to the method of the invention can be made using techniqueswell known to those skilled in the art. For example, any DNA sequenceencoding the antigens can be made using standard techniques and insertedinto any number of expression vectors. Suitable expression vectors havebeen described in the literature and many are commercially available.Likewise, a wide variety of expression systems are known in the art andare commercially available, including prokaryotic and eukaryoticsystems. The proteins can be isolated from the expression systems or anyother suitable source and purified for use in the invention to anydesired degree of purity.

The biological sample obtained from the individual can be any biologicalsample that comprises antibodies, and can comprise biological tissueand/or biological liquid. In various embodiments, the biological liquidis blood, serum or saliva.

The autoantibodies that are positively correlated with SD as describedherein can be detected using any suitable technique, device, systemand/or reagents, many of which are commercially available and/or areotherwise well know to those skilled in the art. In various,embodiments, suitable detection techniques include but are notnecessarily limited to immunohistological techniques, Western blotting,multi-well assay plates adapted for detection of the antibodies, beadsadapted for detection of the antibodies, a lateral flow device or stripthat is adapted for detection of the antibodies, ELISA assays, or anymodification of an ELISA assay that is suitable for detecting theantibodies. Further, any and alls isotypes of the antibodies can bedetected. It is considered that the early antibodies are all orpredominantly IgM and later antibodies are comprised of IgM and IgG.Thus, if desired, the invention can be adapted to discriminate betweenisotypes to assist in determining, for example, the stage of disease.

The method of the invention is suitable for performing on a biologicalsample obtained from an individual of any age or gender. The method maybe performed once, or a series of tests may be performed to, forexample, monitor an individual's response to a treatment.

In one embodiment, the invention is suitable for determining early SD.Early SD is considered to be a stage of SD before salivary and/orlachrymal gland function is diminished to a point where clinicalsymptoms of SD become manifest. Those skilled in the art will be able torecognize early SD, particularly when provided the benefit of thepresent disclosure. Further, the invention provides in some individuals,such as those with other autoimmune diseases or a family history SD,testing for SD before any symptoms appear.

In one embodiment, the invention comprises fixing the result ofperforming the method of the invention in a tangible medium ofexpression, such as a digitized computer record. The invention furthercomprises communication the result of the performing the method of theinvention to a health care provider.

The invention also provides kits comprising the antigens to which theantibodies of SD patients are directed and may further comprisecomponents for biological sample collection and reagents for antibodydetection, positive controls, and the like. In one embodiment, theinvention provides a kit comprising purified SP-1, PSP and CA6 proteinsor fragments thereof that are recognized by antibodies produced by SDpatients. Fragments of these proteins can be recognized by antibodiesproduced by individuals who have SD can be determined using routineskill if given the benefit of the present disclosure. In general, thefragments will be from 9 amino acids in length, up to one amino acidless than the full length the proteins, and including all integers from9 amino acids up to one amino acid less than the full length of theproteins, inclusive. Each and every fragment of these proteins istherefore considered part of the instant disclosure for use in thepresent invention. Each of these fragments can be made and tested todetermine whether antibodies from individuals who have SD recognize thefragments. The proteins or fragments thereof may be provided in isolatedand purified form, and they may be associated with a solid matrix. Thesolid matrix may be present in as a component of any system that can beused for antibody detection, such as multi-well assay plates, beads,lateral flow devices or strips, or any other form or format that issuitable for keeping the proteins in a position whereby antibodies canbind to them and be detected in the method of the invention. Theproteins may be covalently or non-covalently associated with the solidmatrix.

The following Examples are intended to illustrate certain embodiments ofthe invention but are not meant to limit the invention.

EXAMPLE 1

This Example demonstrates the production of autoantibodies in the seraof SD mouse models in the early stages of SD.

In order to obtain the results presented in FIG. 1, sera were collectedfrom IL-14aTG mice, NOD and C57BL/6 mice at 7 months of age. Weexpressed and purified SP-1, PSP and CA6 and used the purified proteinsfor Western blot analysis. Data shown are representative of 6 micestudied in each group. The left panel shows the serum of an IL-14aTGmouse that recognizes CA6 and PSP strongly and SP-1 weakly. The middlepanel shows the same study with the serum of a C57BL/6 mouse. None ofthese auto-antigens were bound by C57BL/6 sera. The right panel showsthe same study with the serum of an NOD mouse. Both CA6 and PSP arestrongly recognized by this serum.

EXAMPLE 2

This Example demonstrates that LTA is found in the salivary glandsecretions of IL-14aTG mice but not in littermate controls.

In order to obtain the data results presented in FIG. 2, salivary glandsecretions were collected from IL-14a TG mice and various control miceat 12 months of age. Western blot assays were performed on the undilutedspecimens. Lanes 1 and 2 are from IL-14a TG mice, lanes 3 and 4 fromIL-14aTG. LTA−/− mice, lanes 5 and 6 from LTA−/− mice and lanes 7 and 8from C57BL/6 mice. We also analyzed the histology of the submandibularand parotid glands in IL-14aTG.LTA−/− mice. These are normal.

EXAMPLE 3

This Example demonstrates that LTA is found in the sera of human SDpatients. In order to obtain our results which are presented in FIG. 3,sera were obtained from 6 normal donors (age and sex matched to 6 of theSjogren's disease patients) and 12 patients with Sjogren's disease.Lymphotoxin levels were determined by a commercially available ELISA(R&D SYSTEMS, Inc). The difference between the serum levels oflymphotoxin between normal controls and patients with Sjogren's diseasewas statistically significant (p=0.0011).

EXAMPLE 4

This Example demonstrates the identification of autoantibodies in thesera and salivary glands of IL-14aTG.LTA−/− mice. The data presented inFIG. 4 show that the serum of an IL-14aTG.LTA−/−mouse at 11 months ofage reacts with SP-1, CA6 and PSP. The Western blot was performed asoutlined in FIG. 1.

EXAMPLE 5

This Example demonstrates that serum obtain from human SD patientscontains antibodies to murine CA6, murine PSP and murine SP-1. Westernblots were performed essentially as described for FIG. 1, except serawere used from patients with SD or aged matched normal controls. Fivepatients and two normal controls were evaluated. Data shown are from onerepresentative patient (all five patients showed similar results) andone normal control (both normal controls showed similar results).Patients with SD but not normal controls have antibodies to PSP andSP-1. The antibody response to CA6 was stronger in SD patients than innormal controls. Thus, we have demonstrated that the presence ofantibodies to CA6, PSP and CA6 can be used to diagnose SD in humans.

While the invention has been shown and described with reference tocertain preferred embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdescribed.

1. A method for determining whether an individual has Sjogren's disease (SD) comprising i) determining in a biological sample from the individual the presence of antibodies directed to salivary gland protein 1 (SP-1), parotid secretory protein (PSP), carbonic anhydrase 6 (CA6), or determining a combination of the antibodies, and determining that the individual has SD based on the presence of the antibodies; or determining in a biological sample obtained from the individual the absence of detection of antibodies to PSP and SP-1 and determining based on the absence of detection of the antibodies that the individual does not have SD.
 2. The method of claim 1, wherein the combination of the antibodies includes antibodies to SP-1.
 3. The method of claim 1, wherein the individual has early SD.
 4. The method of claim 1, wherein the individual is a human.
 5. The method of claim 4, wherein the determining of the antibodies is performed by ELISA assay.
 6. The method of claim 5, wherein the antibodies in the biological sample that are directed to SP-1 bind to murine SP-1 in the ELISA assay.
 7. The method of claim 1, wherein the individual who does not have SD has a lower amount of antibodies to CA6 than an individual who has SD.
 8. The method of claim 1, further comprising determining the presence or absence of antibodies to lymphotoxin (LTA), mucin 10 (submandibular gland salivary mucin), salivary amylase 1, Ro, La, muscarinic receptor 3, fodrin, IL-14, interferon-α, and combinations thereof.
 9. A kit comprising isolated salivary gland protein 1 (SP-1) or a fragment thereof that is recognized by antibodies from an individual with Sjögren's disease (SD), isolated parotid secretory protein (PSP) or a fragment thereof that is recognized by an individual with SD, isolated carbonic anhydrase 6 (CA6) or a fragment thereof that is recognized by antibodies from an individual with SD, or combinations thereof.
 10. The kit of claim 9, wherein the isolated SP-1 or fragment thereof, PSP or fragment thereof, and CA6 or fragment thereof are associated with a solid matrix. 